Reproductive function is tightly regulated by a complex network of central and peripheral factors, where the most important is GnRH. Recently, the neuropeptides kisspeptin (encoded by Kiss1) and neurokinin B (NKB, encoded by Tac3) have been placed as crucial at different stages of reproduction (Navarro V M. Front Endocrinol. 2012; 3:48.). Studies in humans have revealed that loss-of-function mutations in the genes encoding NKB or neurokinin 3 receptor (NK3R) lead to hypogonadotropic hypogonadism and infertility.
Neurokinin B (NKB) is a member of the tachykinin family of peptides. Inactivating mutations in the tachykinin 3 (tac3) or tac3 receptor (NKBR) gene are associated with pubertal failure and congenital hypogonadotrophic hypogonadism in humans. This suggests that NKB may have a critical role in human reproduction.
NKBs have direct action through receptors on the pituitary and indirect through receptors on gonadotropin-releasing-hormone (GnRH) neurons. NKBs bind to their cognate receptors, they stimulate their activity, which in turn provides an obligatory signal for gonadotropin secretion-thus gating down-stream events supporting reproduction. NKB is an important regulator of the hypothalamic-pituitary-gonadal axis and is the target of a range of regulators, such as steroid hormone feedback, nutritional and metabolic regulation.
Energy homeostasis and reproduction are the most important processes in an animal's life and are intimately related. Proper regulation of energy homeostasis and reproduction is fundamental for fitness and survival. Reproduction is an energy-intensive process, and precise interaction of regulators for energy balance and reproduction allows coordinated regulation of these two processes. In most fish species studied, seasonal variations in gonadal size are negatively correlated with serum growth hormone (GH) concentrations—e.g. when luteinizing hormone (LH) concentrations are high, due to gonadal size increase, GH concentrations are low, accompanied by very slow somatic growth.
Fish possess a large diversity of reproduction strategies, can be found in different environmental niches and use different timing regimes of sexual maturation. When compared with other vertebrates, fish have several unique characteristics. In contrast to tetrapod, where the cells in the pituitary are mixed, in fish there is a unique organization of specific calls in certain areas. Fish possesses a dual mode of gonadotrope regulation by GnRH, that combines both neuroglandular and neurovascular components. Moreover, different nerve terminals that secrete different neuropeptides innervate the pituitary. However, it is still unknown whether NKB or NKF neurons project to the pituitary in fish.
To date, a large number of tachykinins have been identified in a wide range of species from invertebrates to mammals Tac1 encodes both substance P (SP) and NKA through alternative splicing. Tac2/Tac3 produces the peptide NKB, and Tac4 encodes hemokinin-1.
Three classes of mammalian tachykinin receptors (NK1, NK2, and NK3) have been identified, and these have preferential binding affinities for SP, NKA, and NKB, respectively. The mammalian TAC1 and TAC4 give rise to 2 active neuropeptides, whereas the TAC3 is the only TAC that give rise to only 1 neuropeptide, namely NKB.
Tachykinin (tac) and tac receptor genes were recently identified from many fish species (Biran 2012, PNAS 109:10269-10274). Phylogenetic analysis showed that piscine Tac3s and mammalian neurokinin genes arise from one lineage. High identity was found among different fish species in the region encoding the NKB; all shared the common C-terminal sequence. Although the piscine Tac3 gene encodes for two putative tachykinin peptides, the mammalian orthologue encodes for only one. The second fish putative peptide, referred to as neurokinin F (NKF), is unique and found to be conserved among all tested fish species.
Zebrafish tac3a mRNA levels gradually increased during the first few weeks of life and peaked at pubescence. In the brain of zebrafish, tac3a and tac3b mRNA was observed in specific brain areas that are related to reproduction (Biran et al., 2008, Biol Reprod 79:776-786). Furthermore, a single ip injection of NKBa or NKF significantly increased LH levels in mature female zebrafish, and the tac3a and both tac3r genes were upregulated by estrogen (Biran et al., 2012, ibid), suggesting that the NKB/NKBR system may participate in neuroendocrine control of fish reproduction and that the role of the NKB system in the neuroendocrine control of reproduction is evolutionarily conserved in vertebrates.
Tilapia have become one of the most commercially important cultured freshwater fish, due to their high growth potential, short generation time, ease of spawning, and disease resistance.
It was shown (Biran et al., 2014, Endocrinology 155, 4831-42) that the recently identified neuropeptides denoted Neurokinin B (NKB) and Neurokinin F (NKF), that are secreted by the fish brain and involved in reproduction, can stimulate the release of follicle stimulating factor (FSH) and LH by direct (through activation of specific receptors at the pituitary level) or indirect (through the brain) mechanisms.
WO 2013/018097, to some of the inventor of the present invention, discloses NKB and NKF agonists for hormonal regulation in fish and specifically for advancing the onset of puberty, regulating the timing and amount of ovulation and spawning, synchronization or stimulation of reproduction, enhancing the development of gammets, enhancing vitellogenesis, induction of GnRH, induction of Kisspeptine, increase in the levels of hypothalamic neurohormones, increasing the level of LH or FSH and induction of oocyte maturation.
G. Drapeau et al., (Regul. Peptides, 31, 125, 1990) discloses the compound SR142801 (Trp7, β-Ala8-Neurokinin A, 4-10) as a potent antagonist of the tachykinin NK3 receptor in mammalians.
O'Harte, F. (J. Neurochem. 57 (6), 2086-2091, 1991) discloses the peptide analog denoted Ranakinin, an NK1 tachykinin receptor agonist isolated with neurokinin B from the brain of the frog Rana ridibunda. 
Several small molecule, non-peptidic NKB antagonists are known in mammals, for example: SB-222200 (Sarau et al., 2000, J Pharmacol Exp Ther 295:373-381); Osanetant (SR-142,801) and talnetant (SB 223412) (Sarau et al 1997, J Pharmacol Exp Ther 281:1303-1311).
While previous publications disclosed fish NKB peptide agonists for enhancing fertilization and shortening the time for maturation or mammalian NK-3 antagonists, none of the prior publications disclose NKB antagonists in fish. There is an unmet need for such antagonists for use in delaying maturation and controlling reproductive parameters in fish.